Targeted modified il-1 family members

ABSTRACT

The present disclosure relates to a modified Interleukin-1 (IL-1) family member cytokine, with reduced activity via its cytokine receptor, wherein said interleukin-1 family member cytokine is specifically delivered to target cells. Preferably, the IL-1 family member cytokine is a mutant, more preferably it is a mutant IL-1 with low affinity to the IL-1 receptor, wherein said mutant IL-1 is specifically delivered to target cells. The targeting is preferably realized by fusion of the modified IL-1 family member cytokine to a targeting moiety, preferably an antibody or antibody-like molecule. The disclosure relates further to the use of such targeted modified IL-1 family member cytokine to treat diseases.

The present invention relates to a modified Interleukin-1 (IL-1) familymember cytokine, with reduced activity via its cytokine receptor,wherein said Interleukin-1 family member cytokine is specificallydelivered to target cells. Preferably, the IL-1 family member cytokineis a mutant, more preferably it is a mutant IL-1 with low affinity tothe IL-1 receptor, wherein said mutant IL-1 is specifically delivered totarget cells. The targeting is preferably realized by fusion of themodified IL-1 family member cytokine to a targeting moiety, preferablyan antibody or antibody-like molecule. The invention relates further tothe use of such targeted modified IL-1 family member cytokine to treatdiseases.

The Interleukin-1 (IL-1) family consists of 11 structurally relatedfamily members (IL-1a, IL-1-(3, IL-1Ra, IL-18, IL-33 and IL-1F5 toIL-1F10), that are among the most potent immune system signalingmolecules, acting through a group of closely related receptors. All IL-1receptors have a similar mode of activation: upon binding of ligand tothe primary receptor subunit (i.e. IL-1R1 for IL-1α and β, IL-18R forIL-18 and ST2 for IL-33), a second receptor subunit is recruited (i.e.IL-1RAP for IL-1α and β, IL-18RAP for IL-18 and IL-1RAP for IL-33) andsignalling is initiated via juxtaposition of the receptor subunits'cytoplasmic Toll/IL-1 receptor (TIR) domains. The dimerized TIR domainsprovide a docking platform for the MYD88 adaptor protein, which viarecruitment of other intermediates leads to activation of thepro-inflammatory nuclear factor-κB (NF-κB) and mitogen-activated proteinkinase (MAPK) pathways. The IL-1 family members are primarily producedby innate immune cells and act on a variety of cell types during theimmune response (for review see Sims and Smith, 2010).

T lymphocytes are one of the main IL-1 family target cells and thepotentiating effects of in particular IL-1α and IL-1β on the expansionand differentiation of different T cell subsets, in particular CD8+ Tcells (Ben-Sasson, 2011; Ben-Sasson, 2013) and Th17 cells (Sutton etal., 2006; Acosta-Rodriguez et al., 2007; Dunne et al., 2010; Shaw etal., 2012) have been firmly established. Th17 cells are characterized bythe production of IL-17 and play an important role in auto-immunedisease and chronic inflammation (reviewed in Wilke et al., 2011). AmongT cell subsets, Th17 cells express the highest levels of the IL-1R andIL-1 plays an important role in Th17 priming.

IL-18 is best known as an IFNγ-inducing cytokine with a potent action onTh1 cells and natural killer (NK) cells, on (Okamura et al., 1995;Takeda et al. 1998). In addition, IL-18 enhances neutrophil function(Leung et al., 2001). Several reports demonstrate IL-18 anti-tumouraction in animal models (Micallef et al., 1997; Loeffler et al., 2008;Wigginton et al., 2002; Zaki et al., 2010) and recombinant human IL-18therapy recently entered clinical trials to evaluate its efficacy fortreatment of advanced cancer (Robertson et al., 2008). As opposed toIL-18, IL-33 acts primarily on Th2 cells (Schmitz et al., 2005) and mastcells (Allakhverdi et al., 2007), and recently was shown to act on CD8+T cells to drive antiviral responses (Bonilla et al., 2012). The otherIL-1 family members are less well characterized, but in summarydifferent IL-1 family members have specificities for different T-cellsubsets or other cell types and hence different therapeuticapplications.

Besides having indirect anti-tumour activity, via activation of T and NKcells, IL-1 family members were shown to have direct cytostaticproperties, which were most convincingly demonstrated on human melanomacells (Morinaga et al., 1990; Usui et al., 1991; Rangnekar et al.,1992).

In view of the contribution of several IL-1 family members toinflammatory processes, clinical interest has been mainly orientedtowards the development of IL-1-antagonizing strategies (Dinarello etal., 2012). Nevertheless, exploitation of controlled agonistic IL-1activity could have applications in different physiological/pathologicalprocesses, where immunostimulatory effects would be desirable. One ofthe main concerns regarding the use of IL-1 in immunostimulatorytherapies, is its severe toxicity when administered systemically.However, when IL-1 action could be confined to a selected cellularpopulation, the toxicity issue might be resolved, which opens uptherapeutic perspectives.

For instance, although there has been a lot of interest on blocking Th17responses in view of their pathogenic role in auto-immune conditionssuch as multiple sclerosis, rheumatoid arthritis and inflammatory boweldisease (Wilke et al., 2011), normal Th17 function is indispensable forprotective immunity against a range of pathogens, includingMycobacterium tuberculosis (Khader et al., 2007), Klebsiella pneumoniae(Ye et al., 2001) and Bordetella pertussis (Higgins et al., 2006). AsIL-1β stimulates Th17 function, the idea has been raised to use IL-1β asa T-cell adjuvant to enhance the response to weak vaccines (Ben-Sassonet al., 2011). Other applications could be the targeting of IL-1β orIL-33 to the CD8+ T-cell population to enhance antiviral responses ortargeting IL-18 to Th1 cells or NK cells to promote anti-tumor activity.

Surprisingly we found that it is possible to design IL-1 familymodifications that are defective in activating their receptor, but, whenfused to a targeting moiety, regain their activity on selected celltypes by a concentration effect at the cell surface. The IL-1 mutantshave a reduced affinity for their cognate receptors, and hence areunable to efficiently bind and activate their receptors. However, byfusing them to a targeting moiety (such as a nanobody) the activity ofthe mutant IL-1 family member is restored on cells expressing the cellsurface target, recognized by the targeting moiety. Because theactivation is confined to the selected targeted cell types only, nomajor systemic toxicity occurs.

A first aspect of the invention is a targeting construct, comprising amodified IL-1 family member cytokine, characterized by a reducedaffinity for its cytokine receptor, and a targeting moiety. IL-1 familymember cytokines are known to the person skilled in the art, andinclude, but are not limited to IL-1α, IL-1β, IL-1Ra, IL18, IL-36Ra,IL-36α, IL-37, IL-36β, IL-36γ, IL-38 and IL-33 (also indicated asIL-1F1, IL-1F2, IL-1F3, IL-1F4, IL-1F5, IL-1F6, IL-1F7, IL-1F8, IL-1F9,IL-1F10 and IL-1F11, respectively). For a review on the IL-1 family, seeDinarello (2011). A modified IL-1 family cytokine means that the IL-1family cytokine has been changed to alter the affinity to its receptor,with as final result that the modified IL-1 family cytokine has areduced affinity for the receptor and a consequent reduced biologicalactivity, as compared to the endogenous wild type cytokine that bindsnormally to the receptor. Such a modification can be a modification thatdecreases the activity of the normal wild type cytokine, or it can be amodification that increases the affinity of a homologous, non-endogenousIL-1 family cytokine (such as, but not limited to a IL-1 family cytokineof another species that is not active on a human IL-1 family cytokinereceptor). Modifications can be any modification reducing or increasingthe activity, known to the person skilled in the art, including but notlimited to chemical and/or enzymatic modifications such as pegylationand glycosylation, fusion to other proteins and mutations. Preferablysaid modification is a mutation, even more preferably it is a mutationdecreasing the affinity of the IL-1 family cytokine. A reduced affinityand a consequent reduced biological activity as used here means that themodified IL-1 family cytokine has a biological activity of less than 70%of the biological activity of the IL-1 family cytokine, even morepreferably less than 60% of the biological activity of the IL-1 familycytokine, more preferably less than 50% of the biological activity ofthe IL-1 family cytokine, more preferably less than 40% of thebiological activity of the IL-1 family cytokine, more preferably lessthan 30% of the biological activity of the IL-1 family cytokine, morepreferably less than 20% of the biological activity of the IL-1 familycytokine, more preferably less than 10% of the biological activity ofthe IL-1 family cytokine, most preferably less than 1% of the biologicalactivity of the IL-1 family cytokine as compared to the IL-1 familycytokine that normally binds to the receptor. Preferably, the modifiedIL-1 family cytokine is a mutant of the wild type IL-1 family cytokineand the activity is compared with the wild type IL-1 family cytokine.The affinity and/or the activity can be measured by any method known tothe person skilled in the art.

A preferred embodiment of the invention is a targeting construct,comprising a mutant IL-1β characterized by reduced affinity for theInterleukin-1 receptor type I (IL-1RI) and/or the interleukin-1 receptoraccessory protein (IL-1RAcP) receptor, and a targeting moiety. A mutantIL-1β as used here can be any mutant form that has a lower affinity forthe receptor and as a consequence a reduced activation of theproinflammatory transcription factor NFκB. The affinity of the mutantIL-1β to the receptor, in comparison to the affinity of the wild typeIL-1β to the receptor can be measured by Scatchard plot analysis andcomputer-fitting of binding data (e.g. Scatchard, 1949) or byreflectometric interference spectroscopy under flow through conditions,as described by Brecht et al. (1993). The activity of the mutant IL-1βis typically measured using a bioassay (for example by the induction ofcell death) or by measuring signaling events downstream of the receptor.Such signaling events can be the modification or nuclear translocationof NF-κB, or the induction of a selected reporter gene. The mutant maybe a point mutant, a deletion or an insertion mutant, or a combinationthereof; several mutations may be present in one protein. Preferably,said mutant IL-1β is obtained by active mutagenesis, such as, but notlimited to site directed mutagenesis by polymerase chain reactionamplification. Preferably, said mutant IL-1β has a biological activityof less than 70% of the biological activity of the wild type IL-1β, evenmore preferably less than 60% of the biological activity of the wildtype IL-1β, more preferably less than 50% of the biological activity ofthe wild IL-1β, more preferably less than 40% of the biological activityof the wild IL-1β, more preferably less than 30% of the biologicalactivity of the wild IL-1β, more preferably less than 20% of thebiological activity of the wild IL-1β, more preferably less than 10% ofthe biological activity of the wild type, most preferably less than 1%of the wild type of which it is deduced (i.e. the wild type IL-1β ofwhich the coding sequence has been mutated to obtain the mutant IL-1β).Preferably, said mutant is a mutant selected from the group consistingof A117G/P118G, R120X, L122A, T125G/L126G, R127G, Q130X, Q131G, K132A,S137G/Q138Y, L145G, H146X, L145A/L147A, Q148X, Q148G/Q150G, Q150G/D151A,M152G, F162A, F162A/Q164E, F166A, Q164E/E167K, N169G/D170G, I172A,V174A, K208E, K209X, K209A/K210A, K219X, E221X, E221S/N224A,N224S/K225S, E244K, N245Q (wherein X can be any change in amino acid,preferably a non-conservative change). Even more preferably saidmutation is selected from the group consisting of R120A, R120G, Q130A,Q130W, H146A, H146G, H146E, H146N, H146R, Q148E, Q148G, Q148L, K209A,K209D, K219S, K219Q, E221S and E221K. Most preferably said mutation isselected from the group consisting of R120G, H146N, H146R, Q148E, Q148Gand K209A. (numbering base on the human IL-1β sequence, genbankaccession number NP_000567, version NP-000567.1, GI: 10835145).

Preferred regions for mutations for IL-18 are Y37-K44, R49-Q54, D59-R63,E67-C74, R80, M87-A97, N127-K129, Q139-M149, K165-K171, R183 andQ190-N191. Most preferred are the regions E67-C74 and M87-A97 (numberingbased on the human sequence, genbank accession number AAV38697, versionAAV38697.1, GI: 54696650).

Preferred regions for mutations for IL-33 are I113-Y122, S127-E139,E144-D157, Y163-M183, E200, Q215, L220-C227 and T260-E269 (numberingbased on the human sequence, genbank accession number NP_254274, versionNP_254274.1, GI:15559209)

Preferably, said targeting moiety is targeting to a marker expressed onan IL-1β receptor expressing cell, preferably a cell expressing IL1-RI.In one preferred embodiment, said targeting moiety is directed to atissue specific marker.

The modified IL-1 family member is linked to a targeting moiety.“Linked” as used here may be by a covalent binding, or it may be by anaffinity binding. A “targeting moiety” as used here is a bindingmolecule that can direct the fusion protein towards a binding site on acell that is expressing a receptor for the IL-1 family member, byspecific interaction between the binding site and the binding molecule.In one preferred embodiment, said binding molecule is a small compound,specifically binding to a molecule situated on the outside of the cell.In another preferred embodiment, said molecule is a sugar structure,directed towards a lectin-like molecule expressed on the cell wall. Inanother preferred embodiment said binding molecule is a peptide,targeting the tumor or inflammation environment. Such peptides are knownto the person skilled in the art, and include, but are not limited toNGR and RGD peptides (Yang et al., 2011; WO2005054293). In still anotherpreferred embodiment, said binding molecule is a protein comprising abinding domain. This includes, but is not limited to carbohydratebinding domains (CBD) (Blake et al, 2006), lectin binding proteins,heavy chain antibodies (hcAb), single domain antibodies (sdAb),minibodies (Tramontano et al., 1994), the variable domain of camelidheavy chain antibodies (VHH), the variable domain of the new antigenreceptors (VNAR), affibodies (Nygren et al., 2008), alphabodies(WO2010066740), designed ankyrin-repeat domains (DARPins) (Stumpp etal., 2008), anticalins (Skerra et al., 2008), knottins (Kolmar et al.,2008) and engineered CH2 domains (nanoantibodies; Dimitrov, 2009).Preferably, said targeting moiety consists of a single polypeptide chainand is not post-translationally modified. Even more preferably, saidtargeting moiety is a nanobody.

The targeting moiety can be any targeting moiety known to the personskilled in the art. In a non-limiting example, said targeting moiety maybe a bispecific antibody, directed to a binding site on the target cellfor one specificity, and to the targeted cytokine, or to a tag fused tosaid cytokine for the other specificity. In another non-limitingexample, the targeting moiety may be chemically linked to the mutantInterleukin-1, or it may be a recombinant fusion protein. Preferably,said targeting construct is a recombinant fusion protein. The targetingmoiety may be fused directly to the mutant IL-1β, or it may be fusedwith the help of a linker fragment, preferably a GGS linker. Thetargeting moiety may be fused at the aminoterminal or at thecarboxyterminal end of the mutated IL-1β; preferably said targetingmoiety is fused at the carboxyterminal extremity of the mutated IL-1βmolecule. The targeting construct may further comprise other domainssuch as, but not limited to a tag sequence, a signal sequence, anothercytokine or an antibody.

Another aspect of the invention is a targeting construct according tothe invention for use as a medicament. One preferred embodiment is atargeting construct according to the invention for use in stimulation ofthe immune response. Indeed, it is know that IL-1 treatment can induceantigen expression on B-cells (Killer et al., 1989); likewise, IL-18treatment is augmenting cellular and humoral immunities (Kinoshita etal., 2011). In a similar way, it has been demonstrated that IL-1 acts onT-cells to enhance the magnitude of in vivo immune responses (Ben-Sassonet al., 2011; Ben Sasson et al., 2013). Therefore, one preferred aspectof the invention is the targeting construct according to the inventionfor use as an adjuvant in vaccination. The targeting construct accordingto the invention is especially interesting in this respect, as thepro-inflammatory effect of normal wild type IL-1 makes the applicationof IL-1 as such impossible.

Still another aspect of the invention is a targeting construct accordingto the invention for use in treatment of cancer. Indeed, Morinaga etal., 1990, Usui et al., 1991 and Rangnekar et al., 1992 have shown thatIL-1 family members do have direct cytostatic properties, which weremost convincingly demonstrated on human melanoma cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the IL-1β-nanobody fusion proteins

FIG. 2: Concentration dependency of the induction of the NFκB activityby wild type and mutant Q148G IL-1 Her2 nanobody fusions (A) and otherselected mutants (B), in mock transfected cells, or cells transfectedwith signaling deficient Her2.

FIG. 3: Effect of wild type and mutant (Q148G, L145A/L147A, F162A/Q164E)IL-1 Her2 nanobody fusions on nuclear translocation of endogenous NF-κBp65 in mock transfected cells, or cells transfected with signalingdeficient Her2.

FIG. 4: Induction of the NFκB activity by wild type and 5 different IL-1mutants, fused to an anti-murine leptin receptor nanobody, on cellsexpressing the murine leptin receptor (mLR) or not (no mLR).

FIG. 5: Concentration dependency of the induction of the NFκB activityby IL1 double mutants fused to the Her2 nanobody in mock transfectedcells, or cells transfected with signaling deficient Her2.

EXAMPLES Materials and Methods to the Examples Cloning of IL-1-NanobodyFusion Proteins.

The 4-10 nanobody directed against the murine leptin receptor isdescribed in Zabeau et al. (2012) and in the patent WO 2006/053883. Theanti-Her2 nanobody 1R59B is described in Vaneycken et al. (2011). Bothnanobodies were cloned with a C-terminal His tag in the pMET7 eukaryoticexpression vector. A codon-optimized sequence encoding the mature IL-1βprotein, preceded by the SigK leader peptide, and equipped with anN-terminal HA tag, was generated via gene synthesis (Invitrogen GeneArt). To generate the IL-1β-nanobody fusion proteins, the IL-1β sequencewas cloned 5′ to the nanobody sequence in pMet7, with a 13×GGS linkerseparating the cytokine and nanobody moieties. (FIG. 1)

IL-1β Mutants.

IL-1β mutants expected to have reduced binding affinity for the IL-1Rwere selected based on literature and analysis of published crystalstructures of human IL-1β complexed with its receptor. Mutations in thehIL-1β moiety were created via site-directed mutagenesis (QuickChange,Stratagene) using the mutagenesis primers as indicated in table I:

TABLE I mutants and primers used Fw primer Rev primer  1 A117G/CCGACTACGCTGGCGGCAGTGACGGTGTCA GCAGTTCAGGCTTCTGACACCGTCACTGC P118GGAAGCCTGAACTGC CGCCAGCGTAGTCGG  2 R120A CTGGCGGCAGCGCCCCTGTCGCTAGCCTGACGCAGGGTGCAGTTCAGGCTAGCGACAGG ACTGCACCCTGCG GGCGCTGCCGCCAG  3 R120GGCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGGG GCACCCTGCGCGCTGCCGC  4 L122A CGCTGGCGGCAGTGCCCCTGTCAGAAGCGCGCTGTCCCGCAGGGTGCAGTTCGCGCTTC GAACTGCACCCTGCGGGACAGCTGACAGGGGCACTGCCGCCAGCG  5 T125G/ CGCCCCTGTCAGAAGCCTGAACTGCGGCGGGCTTTTCTGCTGGCTGTCCCGGCCGCCGC L126G CCGGGACAGCCAGCAGAAAAGCAGTTCAGGCTTCTGACAGGGGCG  6 R127G AGAAGCCTGAACTGCACACTGGGGGACAGCGACCAGGCTTTTCTGCTGGCTGTCCCCCA CAGCAGAAAAGCCTGGTC GTGTGCAGTTCAGGCTTCT  7Q130A CCCTGCGGGACAGCGCGCAGAAAAGCCTGG CCAGGCTTTTCTGCGCGCTGTCCCGCAGG G  8Q130W CTGCACCCTGCGGGACAGCTGGCAGAAAAG GCTCATGACCAGGCTTTTCTGCCAGCTGTCCTGGTCATGAGC CCCGCAGGGTGCAG  9 Q131G CTGCGGGACAGCCAGGGGAAGAGCCTGGTCCGCTCATGACCAGGCTCTTCCCCTGGCTG ATGAGCG TCCCGCAG 10 K132AGCACCCTGCGGGACAGCCAGCAGGCTAGCC GGCCGCTCATGACCAGGCTAGCCTGCTGGTGGTCATGAGCGGCC CTGTCCCGCAGGGTGC 11 S137G/CAGCAGAAAAGCCTGGTCATGGGGTACCCC GCAGTGCCTTCAGCTCGTAGGGGTACCCC Q138YTACGAGCTGAAGGCACTGC ATGACCAGGCTTTTCTGCTG 12 L145GGCCCCTACGAGCTGAAGGCAGGTCATCTGC CCATGTCCTGGCCCTGCAGATGACCTGCCAGGGCCAGGACATGG TTCAGCTCGTAGGGGC 13 H146A CGAGCTGAAGGCACTGGCTCTTCAGGGCCACCATGTCCTGGCCCTGAAGAGCCAGTGCC GGACATGG TTCAGCTCG 14 H146GCCTACGAGCTGAAGGCACTGGGTCTGCAGG CCATGTCCTGGCCCTGCAGACCCAGTGCCGCCAGGACATGG TTCAGCTCGTAGG 15 M146E GCTGAAGGCACTGGAGCTGCAGGGCCAGGCCTGGCCCTGCAGCTCCAGTGCCTTCAGC 16 H146N AGCTGAAGGCACTGAATCTGCAGGGCCAGCTGGCCCTGCAGATTCAGTGCCTTCAGCT 17 H146R CTGAAGGCACTGCGTCTGCAGGGCCAGCTGGCCCTGCAGACGCAGTGCCTTCAG 18 L145A/ GCGGCCCCTACGAGCTGAAGGCAGCGCATGCCATGTCCTGGCCCTGCGCATGCGCTGCC L147A CGCAGGGCCAGGACATGGTTCAGCTCGTAGGGGCCGC 19 Q148E GGCACTGCATCTGGAGGGCCAGGACATATGTCCTGGCCCTCCAGATGCAGTGCC 20 Q148G GAAGGCACTGCATCTGGGTGGCCAGGACATGCTGTTCCATGTCCTGGCCACCCAGATGC GGAACAGC AGTGCCTTC 21 Q148LGCACTGCATCTGCTGGGCCAGGACATG CATGTCCTGGCCCAGCAGATGCAGTGC 22 Q148G/CGAGCTGAAGGCACTGCATCTGGGGGGCGG CCTGCTGTTCCATGTCCCCGCCCCCCAGA Q150GGGACATGGAACAGCAGG TGCAGTGCCTTCAGCTCG 23 Q150G/GCACTGCATCTGCAGGGCGGGGCCATGGAA GCTGAACACGACCTGCTGTTCCATGGCCC D151ACAGCAGGTCGTGTTCAGC CGCCCTGCAGATGCAGTGC 24 M152GGCACTGCATCTGCAGGGCCAGGACGGGGAA GCTCATGCTGAACACCACCTGCTGTTCCCCAGCAGGTGGTGTTCAGCATGAGC CGTCCTGGCCCTGCAGATGCAGTGC 25 F162ACATGGAACAGCAGGTGGTGTTCAGCATGAG GTCGTTGCTTTCCTCGCCCTGCACGGCGCCGCCGTGCAGGGCGAGGAAAGCAACGAC TCATGCTGAACACCACCTGCTGTTCCATG 26 F162A/GCAGGTCGTGTTCAGCATGAGCGCCGTGGA GGATCTTGTCATTGCTTTCCTCGCCCTCC Q164EGGGCGAGGAAAGCAATGACAAGATCC ACGGCGCTCATGCTGAACACGACCTGC 27 F166ACCGACTTCACCATGCAGGCCGTCTCCAGCG CCAGATCTGCTGCCGCCGCTGGAGACGGCGCGGCAGCAGATCTGG CTGCATGGTGAAGTCGG 28 Q164E/GCATGAGCTTCGTGGGGGGCAAGGAAAGCA GGCCACGGGGATCTTGTCATTGCTTTCCT E167KATGACAAGATCCCCGTGGCC TGCCCCCCACGAAGCTCATGC 29 N169G/GCAGGGCGAGGAAAGCGGCGGCAAGATCCC CTTCTCTTTCAGGCCTAGGGCCACGGGGA D170GCGTGGCCCTAGGCCTGAAAGAGAAG TCTTGCCGCCGCTTTCCTCGCCCTGC 30 I172AGAAAGCAACGACAAGGCCCCCGTGGCCCTG CCCAGGGCCACGGGGGCCTTGTCGTTGCT GG TTC 31V174A GCAACGACAAGATCCCCGCGGCCCTGGGCC CTTTCAGGCCCAGGGCCGCGGGGATCTTGTGAAAG TCGTTGC 32 K208E GCAGCTGGAAAGCGTGGATCCCAAGAACTAGCGTTTTTCCATCTTTTTCTCGGGGTAGT CCCCGAGAAAAAGATGGAAAAACGCTCTTGGGATCCACGCTTTCCAGCTGC 33 K209A CCCCAAGAACTACCCCAAGGCAAAGATGGAGTTGAACACGAAGCGCTTTTCCATCTTTG AAAGCGCTTCGTGTTCAAC CCTTGGGGTAGTTCTTGGGG34 K209D GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCATCTTGTCCTTGGGGTAGTCCCCAAGGACAAGATGGAAAAACGC TCTTGGGATCCACGCTTTCCAGCTGC 35 K209A/CCCCAAGAACTACCCCAAGGCAGCGATGGA GAACACGAAGCGTTTTTCCATCGCTGCCT K210AAAAACGCTTCGTGTTC TGGGGTAGTTCTTGGGG 36 K219SAAAAACGCTTCGTGTTCAACAGCATCGAGA GAGCTTGTTGTTGATCTCGATGCTGTTGATCAACAACAAGCTC ACACGAAGCGTTTTT 37 K219Q AAAAACGCTTCGTGTTCAACCAGATCGAGACTTGTTGTTGATCTCGATCTGGTTGAACA TCAACAACAAG CGAAGCGTTTTT 38 E221SGCTTCGTGTTCAACAAGATCTCGATCAACA ACTCGAGCTTGTTGTTGATCGAGATCTTGACAAGCTCGAGT TTGAACACGAAGC 39 E221K CTTCGTGTTCAACAAGATCAAGATCAACAATCGAGCTTGTTGTTGATCTTGATCTTGTT CAAGCTCGA GAACACGAAG 40 K219S/GGAAAAACGCTTCGTCTTCAACAGCATCTC CGAACTCGAGCTTGTTGTTGATCGAGATG E221SGATCAACAACAAGCTCGAGTTCG CTGTTGAAGACGAAGCGTTTTTCC 41 E221S/CGCTTCGTGTTCAACAAGATCTCGATCAAC CTCGAACTCGAGCTTGGCGTTGATCGAGA N224AGCCAAGCTCGAGTTCGAG TCTTGTTGAACACGAAGCG 42 N224S/CAACAAGATCGAGATCAACAGCAGCCTCGA CTGGGCGCTCTCGAATTCGAGGCTGCTGT K225SATTCGAGAGCGCCCAG TGATCTCGATCTTGTTG 43 E244KCCCCAACTGGTACATCAGTACTAGTCAGGC GGAACACGGGCATATTCTTGGCCTGACTACAAGAATATGCCCGTGTTCC GTACTGATGTACCAGTTGGGG 44 N245QCAGCACTAGTCAGGCCGAGCAGATGCCCGT GGTGCCGCCCAGGAAGACGGGCATCTGCTCTTCCTGGGCGGCACC CGGCCTGACTAGTGCTG 45 E244K/CATCAGCACTAGTCAGGCCAAGCAGATGCC GGTGCCGCCCAGGAAGACGGGCATCTGCT N245QCGTCTTCCTGGGCGGCACC TGGCCTGACTAGTGCTGATG 46* R120G/GCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGGG Q131GGCACCCTGC GCGCTGCCGC 47* R120G/ CGAGCTGAAGGCACTGGCTCTTCAGGGCCACCATGTCCTGGCCCTGAAGAGCCAGTGCC H146A GGACATGG TTCAGCTCG 49* R120G/GCGGCCCCTACGAGCTGAAGGCAGCGCATG CCATGTCCTGGCCCTGCGCATGCGCTGCC L145A/CGCAGGGCCAGGACATGG TTCAGCTCGTAGGGGCCGC L147A 48** R120G/GCGGCAGCGCCCCTGTCGGAAGCTTGAACT GCAGGGTGCAGTTCAAGCTTCCGACAGGG Q148GGCACCCTGC GCGCTGCCGC 50* R120G/ GCAGGTCGTGTTCAGCATGAGCGCCGTGGAGGATCTTGTCATTGCTTTCCTCGCCCTCC F162A/ GGGCGAGGAAAGCAATGACAAGATCCACGGCGCTCATGCTGAACACGACCTG Q164E C 51* R120G/GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCATCTTTTTCTCGGGGTAGT K208ECCCCGAGAAAAAGATGGAAAAACGC TCTTGGGATCCACGCTTTCCAGCTGC 52** Q131G/CTGCGGGACAGCCAGGGGAAGAGCCTGGTC CGCTCATGACCAGGCTCTTCCCCTGGCTG Q148GATGAGCG TCCCGCAG 53** Q148G/ GCAGGTCGTGTTCAGCATGAGCGCCGTGGAGGATCTTGTCATTGCTTTCCTCGCCCTCC F162A/ GGGCGAGGAAAGCAATGACAAGATCCACGGCGCTCATGCTGAACACGACCTG Q164E C 54** Q148G/GCAGCTGGAAAGCGTGGATCCCAAGAACTA GCGTTTTTCCATCTTTTTCTCGGGGTAGT K208ECCCCGAGAAAAAGATGGAAAAACGC TCTTGGGATCCACGCTTTCCAGCTGC*double/triple-mutants were created using R120G as template.**double/triple-mutants were created using Q148G as template.

Production of IL-1β Fusion Proteins.

IL-1β fusion proteins were produced in HEK293T cells. For small-scaleproduction, HEK293T cells were seeded in 6-well plates at 400000cells/well in DMEM supplemented with 10% FCS. After 24 hours, culturemedium was replaced by medium with reduced serum (DMEM/5% FCS) and cellswere transfected using linear PEI. Briefly, PEI transfection mix wasprepared by combining 1 μg expression vector with 5 μg PEI in 160 μlDMEM, incubated for 10 minutes at RT and added to the wells dropwise.After 24 hours, transfected cells were washed with DMEM and layered with1.5 ml OptiMem/well for protein production. Conditioned media wererecuperated after 48 hours, filtered through 0.45μ filters and stored at−20° C. IL-1β content in the conditioned media was determined by Elisaaccording to the manufacturer's instructions (R&D Systems).

NF-κB Reporter Gene Assay.

To assess IL-1R activation, we used HEK-Blue™ IL-1β cells that stablyexpress the IL-1R (Invivogen) and transfected them transiently with anNF-κB luciferase reporter gene. Briefly, HEK-Blue™ IL-1β cells wereseeded in culture medium (DMEM/10% FCS) in 96-well plates (10000cells/well) and transfected the next day using the calcium phosphateprecipitation method with the indicated amounts of expression plasmidsand 5 ng/well of the 3 KB-Luc reporter gene plasmid (Vanden Berghe etal., 1998). 24 hours post-transfection, culture medium was replaced bystarvation medium (DMEM) and 48 hours post-transfection, cells wereinduced for 6 hours with fusion proteins. After induction, cells werelysed and luciferase activity in lysates was determined using thePromega Firefly Luciferase Assay System on a Berthold centro LB960luminometer.

Analysis of NF-κB Nuclear Translocation Via Confocal Microscopy.

For confocal imaging, 10⁵ HEK293-T cells/well (in 6-well plate) wereseeded on glass coverslips (Zeiss), coated with poly-L-lysine (Sigma).The next day, cells were transfected with 200 ng/well of empty vector orHER2Δcyt expression plasmid using the calcium phosphate precipitationmethod. After 48 hours, cells were treated for 30 minutes with vehicle(medium) or IL1-Her2 nanobody fusion protein (10 ng/ml). Next, cellswere rinsed with 1×PBS and fixed for 15 minutes at room temperature in4% paraformaldehyde. After three washes with 1×PBS, cells werepermeabilized with 0.1% Triton X-100 in 1×PBS for 10 minutes and blockedin 1% BSA in 1×PBS for another 10 minutes at room temperature. Sampleswere then incubated for 1 hour at 37° C. with rabbit anti-p65 antibody(Santa Cruz C20, diluted 1:800) and mouse anti-Flag Antibody (Sigma M2,1:2000). After four washes in 1×PBS, cells were incubated for 1 hour atroom temperature with anti-rabbit Alexa 488 and anti-mouse Alexa 594fluorochrome-conjugated secondary antibodies (both diluted 1:800). Aftersecondary antibody incubation, cells were washed four times in 1×PBS andnuclei were stained with DAPI (2 μg/ml). After a final wash step in1×PBS, coverslips were mounted using propyl gallate. Images wereacquired using a 60×1.35 NA objective on an Olympus IX-81 laser scanningconfocal microscope and analyzed using Fluoview 1000 software.

Example 1: IL-1β-Ligand and IL-1β-Nanobody Fusion Proteins

FIG. 1 shows a scheme of the IL-1β-nanobody fusion proteins constructedwith either WT hIL-1β or the hIL1β mutants described in table I.

Example 2: IL-1β Activity of Selected Mutant IL-1β-Nanobody Fusions isRestored on Cells Expressing the Nb Targets

Wild type IL-1β and 45 IL-1β mutants (Table I) were fused to awell-characterized nanobody recognizing Her2 (1R59B). The IL-1β-nanobodyfusion proteins were tested on HEK-Blue™ IL-1β cells, transientlytransfected with an NF-κB reporter gene plasmid (5 ng/well) and aHer2Δcyt (signalling-deficient) expression plasmid (2 ng/well). Cellswere treated for 6 hours with IL-1β-Her2 nanobody fusions (dose responseranging from 0.4 to 250 ng/ml). As demonstrated in FIG. 2A, theIL-1β-Q148G-Her2 nanobody fusion displayed a reduced ability to activateNF-κB as compared to the WT IL1-β-Her2 nanobody fusion. Importantly,targeting of the Q148G mutant to Her2Δcyt-expressing cells restored itsactivity and produced a dose-response curve for NF-κB activation thatperfectly parallels that of the WT IL-1β on mock-transfected cells. Alsoevident from this figure is a strong targeting effect for the WT IL-1βHer2 nanobody fusion. Similar “activation by targeting” effects wereobserved for six other IL-1β mutants (R120G, Q131G, H146A, H145A/L147A,F162A/Q164E and K208E) fused to the Her2 nanobody (FIG. 2B).

To obtain further proof for the “activation by targeting” concept, wenext explored whether we could visualize the selective activation ofNF-κB in Her2-expressing cells by the IL-1β-Her2 nanobody fusions viaconfocal microscopy. We measured activation of endogenous NF-κB byassaying its nuclear translocation. As evident from FIG. 3, only the WTIL-1β-Her2 nanobody fusion promoted translocation of endogenous NF-κB incells that do not express Her2. Whereas they did not promote detectableNF-κB translocation in mock-transfected cells, the three tested mutantIL1-β-Her2 nanobody fusions triggered NF-κB nuclear translocation incells that also stained positive for Her2, indicating they only act ontargeted cells.

To evaluate whether the “activation by targeting” concept also worksusing a nanobody to an unrelated membrane protein, we fused WT IL-1β andfive of the disabled IL-1β mutants (R120G, Q131G, H146A, Q148G, K209A)to a previously characterized nanobody recognizing the mLR (4-10). Anexperiment similar to that reported for the IL-1β-Her2 nanobody fusion(FIG. 2) was performed using HEK-Blue™ IL-1β cells, transientlytransfected with a mLR expression plasmid (10 ng/well). Similar to theresults obtained with the Her2 nanobody fusion proteins, allinvestigated mutant IL-1β nanobody fusions (tested at 12.5 ng/ml) showeda reduced ability, as compared to the WT fusion, to activate NF-κB oncells that do not express mLRs. However, targeting by the mLR nanobodymoiety partially restored the activity of the selected mutants (FIG. 4).

Because the IL-1β mutants described above retained significant residualbiological activity, we combined different mutations to obtaindouble/triple mutants with reduced basal activity. Nine double/triplemutants were tested (cf. table I mutants 46 to 54) and from these, sixmutant proteins (Q131G/Q148G, Q148G/K208E, R120G/Q131G, R120G/Q131G,R120G/H146A, R120G/K208E, R120G/F162A/Q164E) displayed no residualactivity (using the same assay for measuring NF-κB as in FIG. 2) onHer2-negative cells, whilst partially restored activity was apparent oncells overexpressing Her2Δcyt (FIG. 5).

These data altogether indicate that targeting partially inactive mutantIL-1β, by fusing it to a nanobody recognizing a cell surface receptor,can restore its activity on nanobody target cells, probably by forcedreceptor interaction through a membrane concentration effect. The factthat activation by targeting can be accomplished using nanobodiesrecognizing different classes of membrane proteins indicates broadapplicability of the “activation by targeting” concept.

Because these data provide proof of concept for the ability of targetingmutant IL-1 family members to selected cell types, restoring theiractivity on these target cells only, nanobodies are produced that allowtargeting IL-1 family members to physiologically relevant IL-1β targetcells. In view of the important role of IL-1 family members as T- andNK-cell activators, the nanobodies are designed to specifically targetIL-1 to T- and NK-cell subsets. More specifically nanobodies targetingCCR6, which are predominantly expressed on Th17 cells as well asnanobodies targeting CD8 on cytotoxic T cells are developed and fused tothe members of the IL1-family, preferably IL-1β.

Example 3: Effect of IL-1β-Nanobody Fusions on IL-17 Production byPrimary Human T Cells

Primary human T cells were isolated from buffy coats. First, PBMC's wereisolated by lymphoprep density gradient centrifugation and incubated 0/Nwith 0.5 ng/ml rhIL-2 for recovery. Next, T-cells were isolated usingthe pan-T cell isolation kit (Miltenyi Biotec) according to themanufacturer's instructions. Briefly, T cells were resuspended(1×10⁶/ml) in RPMI-1640 supplemented with 10% FCS and CD3/CD28activating microbeads (Miltenyi Biotec). Next, cells (100 μl/well) wereplated in U-bottom 96-well plates and stimulated for 96 hours with theindicated concentrations of IL-1β variants. After an additional 6 hoursstimulation with PMA/ionomycin (both at 100 nM), supernatants wererecovered and IL-17 levels were determined by Elisa (R&D Systems).Additional cytokines are evaluated via Luminex technology.

For selected mutant IL-1β-nanobody fusions (e.g. with a nanobodytargeting CCR6) target cell-specific IL-17 and IFNγ production areevaluated by intracellular staining using a flow cytometric approach.

Also, to corroborate selectivity for the Th17 population, binding toPBMC subpopulations is measured via double staining using the Flag tagand selected CD markers, followed by flow cytometric analysis.

Finally, in a clinically relevant in vitro model of human Th17 cellfunction, the adjuvant activity of the IL-1β-nanobody fusions isassessed. In view of the need for more efficacious vaccines againstBordetella pertussis (or adjuvants for the existing vaccines), wedetermined whether the selected fusion proteins enhance the human Th17response in a coculture model of naïve T cells with B. pertussis-treatedmonocyte-derived dendritic cells (MDDCs). Human MDDCs are isolated frombuffy coats (using the monocyte isolation kit II, Miltenyi Biotec),treated with different ratios of B. pertussis for 48 hours and thencocultured with naïve allogeneic T cells for 12 days. Afterrestimulation with anti-CD3/anti-CD28, the cytokine profiles insupernatants are determined using Elisa/Luminex technology (cfr. supra).

Example 4: Effect of IL-1β-Nanobody Fusions on CTLs

To assess whether IL-1β-CD8 nanobody fusions can specifically enhancethe function of CD8+ T cells, human PBMC's are isolated by lymphoprepdensity gradient centrifugation from buffy coats and stimulated for 24hours with CD3/CD28 activating microbeads (Miltenyi Biotec) incombination with wt or mutant IL1β-CD8 Nb fusions. The effect of thesefusion proteins on CD8+ T cell activation is evaluated by performingintracellular staining for active (phosphorylated) NF-κB and IFNγ. Inaddition, to investigate whether the IL-1β-nanobody fusions affect CTLdegranulation, PBMC's (2×10⁶ cells/ml) are differentiated for 48 hoursin the presence of phytohaemagglutinin (PHA, 1 μg/ml) and IL-2 (100IU/ml) in combination with increasing doses of the IL-1β fusionproteins. Next, to induce degranulation, cells are stimulated for 3hours with CD3/CD28 dynabeads and analysed by flow cytometry.Degranulation is measured via detection of cell surface CD107a, awell-established marker for natural killer activity. In all flowcytometric analyses on leukocyte pools, anti-CD8 staining is included toallow monitoring of the cell-type specificity of the IL-1β-CD8 Nbeffects.

Finally to assess whether the IL-1β-CD8 nanobody fusions promoteanti-tumor activity in vivo, C57BL/6 mice are injected subcutaneouslywith TC1 tumor cells, which produce the E6 and E7 antigenic oncoproteinsfrom HPV16. This model was previously used to demonstrate that IL-1βpromotes CD8+ T cell-mediated, antigen-specific, anti-tumor responses(Ben-Sasson, 2013). Briefly, mice are immunized four days after tumorinjection with a vaccine containing the HPV16E7₄₉₋₅₇ peptide, combinedwith DOTAP and LPS, and with our without WT or mutant IL-1β-CD8 Nbfusions or IL-1β-GFP Nb fusions. Tumor size is monitored for 18 dayspost-immunization.

Example 5: In Vivo Experiments—Vaccine Adjuvans Effect

In a first series of experiments C57BL/6 mice are treated iv/ip withdifferent doses of WT and mutant IL-1β-nanobody fusions and unfusedIL-1β, to monitor acute toxicity. Venous blood is collected at differenttimes post treatment by tail venopuncture and the cytokine profile inserum is determined by Luminex assay. In addition, via flow cytometricanalysis intracellular cytokine levels (IL-17, IFNγ) and activation ofIL-1R (as assessed by measuring phospho-NF-κB levels) are determined inselected leukocyte subsets.

When optimal doses have been established, their adjuvant activity isassessed in a murine vaccination protocol. Briefly, C57BL/6 mice areimmunized ip with acellular pertussis vaccine (Pa). The Pa vaccine iscomposed of 5 μg/mouse of purified recombinant detoxified pertussistoxin (PT9K/129G)+filamentous hemagglutinin (FHA) (composition accordingto Brereton et al., 2011). 24 hours after immunization, selected mutantIL1β-Nb or PBS are administered ip or iv. Animals are boosted after 28days. One set of animals is sacrificed 14 days after the secondimmunization and splenocytes are isolated and restimulated in vitro withmedium or FHA for 3 days. Cytokine levels in culture supernatants(IL-17, IFNγ, IL-2, IL-10, IL-5, IL-4, etc.) are determined via Luminextechnology. A second set of mice is challenged with B. pertussis on day14 post-boost and sacrificed 2 h and 5 and 10 days post-challenge. Lungsare isolated and CFU in lung homogenates will be quantified onBordet-Gengou agar plates. Cytokine levels in lung homogenates aredetermined as in splenocyte supernatants.

In addition, blood is sampled (from the tail vene) before immunizationand then every 14 days for determination of B. pertussis-specific IgGlevels in serum.

Example 6: Direct Antitumor Effect of IL-1β-Nanobody Fusions

To investigate the direct anti-tumour activity of selected IL1-nanobodyfusions, we use human A375 melanoma cells, which were shown to be highlysusceptible to IL-1-induced cytostatic effects (Morinaga et al., 1990).To allow targeting of mutant IL-1 family members to the A375 cells, astable A375 clone expressing a cell surface marker to whichhigh-affinity nanobodies are already available (i.e. CD20) is generated.The sensitivity of this cell line, as compared to the parental A375cells, to the antiproliferative effect of the mutant IL1-nanobodyfusion, is investigated in vitro using the XTT proliferation assay. Invivo anti-tumour activity of the mutant IL-1-nanobody fusions isinvestigated using an A375 xenotransplant model. Briefly, athymic nudemice are inoculated subcutaneously with A375 cells (parental orexpressing a surface marker for targeting) and tumor growth is monitoredfor four weeks in animals treated with PBS or mutant IL1-nanobodyfusions.

Example 7: Extension of Principle to IL18: Application in Tumor Models

To assess the indirect anti-tumour activity of IL1 family members,experiments are conducted to address the efficacy of selected mutantIL-18-nanobody fusions using the Meth A syngeneic mouse sarcoma modelaccording to the protocol that was used previously to demonstrateanti-tumour activity of IL-18 (Micallef et al., 1997). IL18 variantsused in these experiments consist of mutant IL-18s fused to nanobodiestargeting immune cells with tumoricidal properties (i.e. CTLs,NK-cells). The mice are treated with the construct, and a significantreduction of the tumor is noted when compared to the mock treatedcontrol.

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1-13. (canceled)
 14. A composition comprising a construct comprising:(1) a mutated human IL-1β cytokine characterized by a reduced activityas compared to wild type human IL-1β cytokine, wherein the mutation isone or more mutations selected from R120G, Q131G, L145A, H146A, L147A,Q148G, F162A, Q164E, and K208E, and the numbering is based on the humanIL-1β sequence, and (2) a targeting moiety directed against human CD8,wherein the targeting moiety restores activity of the mutated humanIL-1β cytokine on target cells.
 15. The composition of claim 14, whereinthe targeting moiety is directed to CD8 expressed on human IL-1βcytokine receptor expressing cell.
 16. The composition of claim 14,wherein the targeting moiety is directed to CD8 expressed on an IL-1R1and/or IL-1RacP expressing cell.
 17. The composition of claim 14,wherein the mutated human IL-1β further comprises at least one mutationfrom mutations comprising A117G/P118G, L122A, T125G/L126G, R127G, K132A,S137G/Q138Y, Q148G/Q150G, Q150G/D151A, M152G, F162A/Q164E, F166A,Q164E/E167K, N169G/D170G, I172A, V174A, K209A, K209A/K210A, E221K,E221S/N224A, N224S/K225S, E244K, or N245Q.
 18. The composition of claim14, wherein the mutated human IL-1β comprises Q131G and Q148G.
 19. Thecomposition of claim 14, wherein the mutated human IL-1β comprises Q148Gand K208E.
 20. The composition of claim 14, wherein the mutated humanIL-1β comprises R120G and Q131G.
 21. The composition of claim 14,wherein the mutated human IL-1β comprises R120G and H146A.
 22. Thecomposition of claim 14, wherein the mutated human IL-1β comprises R120Gand K208E.
 23. The composition of claim 14, wherein the mutated humanIL-1β comprises R120G, F162A, and Q164E.
 24. A method of stimulating animmune response in a cell, comprising contacting the cell with thecomposition of claim 14.